Influenza A virus causes one of the most wide-spread infections in humans. The threat of a flu pandemic looms with the growing number of avian influenza outbreaks and deaths. Given this threat and the drawbacks of existing vaccines and antivirals, the development of new influenza therapies is greatly needed. The National Institutes of Health has therefore designated influenza infection research, including therapeutics research, as a priority area for biodefense research. RNA interference (RNAi) is a process by which double-stranded RNA directs sequence- specific degradation of messenger RNA. We hypothesize that siRNAs specific for conserved regions in the influenza genome will be potent antivirals that will broadly target flu strains and have reduced susceptibility to viral resistance. We further hypothesize that siRNA administered intranasally can be taken up by cells in the lungs and that the efficiency of delivery can be enhanced by the use of carriers. To realize our long term objective of developing prophylactic and therapeutic siRNAs that yield an effective treatment for influenza with a greatly reduced susceptibility to viral resistance, we propose i) to characterize emergence of viral resistance to influenza-specific siRNAs and to study the resistance mechanism, ii) to study the distribution, degradation and cellular uptake of naked siRNA following intranasal delivery to lung, iii) to identify polymers that protect siRNA from degradation and facilitate siRNA lung delivery. In Specific Aim #1, we will investigate in vitro viral resistance induced by individual siRNAs as well as siRNA combinations and compare the best siRNA combination with the neuraminidase inhibitors. We will also test whether siRNAs inhibit viruses that are resistant to the NA inhibitors. In Specific Aim #2 we will determine the tissue distribution of administered siRNA, the specific lung cell types that take up siRNA, and assess the degradation of naked siRNA following intranasal administration. In order to improve the efficiency of siRNA delivery and cell uptake, in Specific Aim #3 we will select up to 10 polymers with known toxicity and disposition that have been used in the clinic, and examine their ability to protect siRNA and facilitate its uptake. It is anticipated that these studies will yield siRNA combinations that are resistant to viral mutations as well as lead siRNA formulations for further study. Progress on this project will significantly advance generation of siRNA flu therapy and, pending successful completion, IND enabling studies of the chosen siRNA combination and delivery method. It is anticipated that Phase I/II clinical studies will follow, ultimately testing an siRNA agent for influenza prophylaxis and therapy, realizing the NIAID's goal of supporting "research on influenza ... to develop new or improved ... treatments..." Tools and knowledge gained from this proposal will also enhance basic understanding of the biology of influenza infection and mechanisms underlying viral resistance to siRNA, as well as advance methodology for nucleic acid delivery to lung. [unreadable] [unreadable] [unreadable]